Session 11: Medical Biotechnology in Regenerative Medicine ... · Paula Mazurek1, Tomasz Pieczonka...

BIO2018 Congress and 51st Meeting of the Polish Biochemical Society

Session 11: Medical Biotechnology in Regenerative Medicine and Drug Research

Lectures

L11.1

Targeting inflammation for treatment of Duchenne muscular dystrophy Magdalena Kozakowska, Olga Mucha, Paulina Podkalicka, Katarzyna Pietraszek-Gremplewicz, Iwona Bronisz-Budzyńska, Alicja Józkowicz, Agnieszka Łoboda, Józef Dulak

Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, PolandJózef Dulak <[email protected]>

Muscle damage in Duchenne muscular dystrophy (DMD), the incurable diseases caused by the lack of dystrophin, is strongly linked to inflammatory reactions. Accordingly, modulation of inflammation can ameliorate the disease progression.In our recent studies we have generated the double knock-out (dKO) mdx mice lacking additionally either the active heme oxygenase-1 gene (HO-1, Hmox1) or miR-378a cod-ing sequence. The HO-1 is the crucial anti-inflammatory protein affecting also muscle stem (satellite) cells differen-tiation, while miR-378a is involved in regulation of muscle differentiation and cellular metabolism.Lack of HO-1 aggravates the severity of DMD in a mdx mice and Hmox1-/-mdx dKO animals demonstrate strongly impaired exercise capacity. Interestingly, mdx satellite cells (SCs) show disturbed and enhanced differentiation, which is further aggravated by Hmox1 deficiency. Interestingly, mdx SCs expressed lower level of Hmox1, and RNA se-quencing demonstrated the significant changes in mdx SCs transcriptome. Importantly, restoration of Hmox1 expres-sion normalized the SCs differentiation.Surprisingly, mice lacking miR-378a were able to run longer distance than their wild-type (WT) in a treadmill test. Lack of miR-378a ameliorated the impairment characteristic for mdx mice, as 3-months old dKO animals were stronger than mdx mice and ran similar distance to WT. The muscles of dKO animals showed less infiltration of inflammatory cells. RNA-sequencing revealed the fibroblast growth fac-tor 1 (Fgf1) among the most downregulated genes in ani-mals lacking miR-378-/- and in dKO mice, what might be of relevance to lower fibrosis observed in dKO than mdx miceIn summary, HO-1 and miR-378a can be considered as tar-gets to alleviate muscle injury and DMD severity.

Acknowledgements:Supported by grants No. 2012/06/A/NZ1/0004 and 2016/21/B/NZ1/00293 from National Science Centre.

L11.2

HO-1 as a possible drug targetPaulina Podkalicka1*, Olga Mucha1*, Szymon Barwacz1, Kalina Andrysiak1, Anna Biela1, Szczepan Kruczek1, Damian Ryszawy2, Maciej Mikulski3, Kamil Sitarz3, Michal Galezowski3, Arkadiusz Bialas3, Tomasz Rzymski3, Krzysztof Brzozka3, Alicja Jozkowicz1, Jozef Dulak1, Agnieszka Loboda1

1Department of Medical Biotechnology, 2Department of Cell Biology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland; 3Selvita S.A., Poland*Equally contributed as the first author

Agnieszka Loboda <[email protected]>

Heme oxygenase-1 (HO-1), a cytoprotective, pro-angi-ogenic and anti-apoptotic enzyme is highly expressed in various cancer types what in turn correlates with the more advanced tumor development as well as poorer survival of patients. Moreover, HO-1 expression is further induced upon chemo-, radio- and photodynamic therapy, what re-sults in decreased effectiveness of the treatment.On the basis of its cytoprotective features, scientists have pursued the targeting of HO-1 as an attractive target for anticancer treatment. The genetic inhibition of HO-1 by RNA interference (RNAi) or CRISPR/Cas9 approaches provides the possibility to specifically target HO-1; howev-er, the potential therapeutic application of those methods are distant at best. Possibilities of specific pharmacological inhibition of HO-1 are strongly limited as commercially available metalloporphyrins exert many unspecific effects. Therefore, we initiated a drug discovery program aimed at the identification and characterization of novel small-molecule inhibitors of HO-1. Virtual High Throughput Screening (vHTS) followed by structure-based rational de-sign led to the identification of molecules which actively inhibit the catalytic function of HO-1 at submicromolar concentrations. The potent anti-cancer activity of analyzed compounds in vitro was demonstrated using broad spec-trum of methods, but in vivo experiments are required to fully establish potential application of small inhibitors of HO-1 as anti-cancer drugs.In summary, HO-1 seems to be an attractive cellular path-way for drug discovery and genetic manipulation, however the ideal strategy for HO-1 targeting requires further stud-ies.

Acknowledgements:Supported by grant no. 2016/21/B/NZ1/00293 from National Science Centre and grant no. PBS2/B7/15/2014 from the National Centre for Research and Development (NCBR).

Session 11: Medical Biotechnology in Regenerative Medicine and Drug Research 65

Gdańsk, September 18th–21st 2018

Oral presentations

O11.1

Regulation of hair follicle Stem Cells homeostasis by activation of gene inhibitory networkXiaoyang Wang2*, Łukasz Boryń1*, Patrycja Daszczuk1, Paula Mazurek1, Tomasz Pieczonka1, Guangfang Wang2, Raul Ramos2, Xiaojie Wang2, Maksim Plikus2, Kyuson Yun3, Mark Israel4, Eve Kandyba5, Krzysztof Kobielak1

1University of Warsaw, Centre of New Technologies, Warsaw, Poland, 2University of California Irvine, Stem Cell Research Center, Irvine, CA, USA, 3The Jackson Laboratory, Bar Harbor, ME, USA, 4Dartmouth Hitchcock Medical Center, Lebanon, NH, USA, 5University of California San Francisco, USA*Co-first authors

Krzysztof Kobielak <[email protected]>

Previously, we revealed how the underlying molecular mechanism of Bone Morphogenetic Protein (BMP) signal-ing governs the homeostasis of hair follicle stem cells (hf-SCs) in vivo. Here, we focused on testing role of Id2 gene, one of the target genes which we identified to be down-regulated in hfSCs after inhibition of BMP pathway. To test the function of Id2 gene in hfSCs in vivo we used Id2 gain of function approach (GOF) by generating transgenic mouse line. Our data demonstrated that Id2 overexpres-sion in hfSCs results in prolonged telogen and a delay in anagen activation, maintaining stem cells quiescence. Fur-ther, we performed FACS sorting and RNA-seq analysis of GOF-Id2 hfSCs at first postnatal hair cycle. By compari-son with common signature genes in quiescent bulge we demonstrated that approximately 10% of the genes were affected by overexpression of Id2 in hfSCs. Interestingly approximately half of those genes overlapped with com-mon signature genes which we previously published to be affected by BMP pathway, thus working synergistically with that pathway. On the other hand our data also suggest that although Id2 is a direct target and an effector of BMP pathway in hfSCs in vivo, second half of those genes work at least partially independent from BMP signaling on gene regulatory network of quiescent bulge.

O11.2

Administration of HO-1/SDF-1α-overexpressing mesenchymal stromal cells in murine and swine model of myocardial infarction and heart failure – analysis of therapeutic effectJacek Stępniewski1, Joanna Śliwka2, Urszula Florczyk1, Mateusz Tomczyk1, Agnieszka Langrzyk3, Ewa Wiśniewska3, Klaudia Kulik3, Marta Głowacka3, Tomasz Jaźwiec2, Marcin Garbacz2, Tomasz Niklewski2, Alicja Józkowicz1, Agnieszka Jaźwa1, Marian Zembala2, Michał Zembala2, Józef Dulak1,3

1Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland; 2Silesian Centre for Cardiac Diseases, Zabrze, Poland; 3Kardio-Med Silesia, Zabrze, PolandJacek Stępniewski <[email protected]>

Objective: Administration of mesenchymal stromal cells (MSC) have been proposed as an approach to regenerate damaged myocardium. The survival of cells upon adminis-tration into infarcted tissue, however, is limited. Therefore, the aim of this study was to evaluate the therapeutic po-tential of mesenchymal cells overexpressing proangiogenic (SDF-1α) as well as anti-inflammatory [heme oxygenase-1 (HO-1)] factors in murine and swine model of myocardial infarction (MI). Methods: In mice (immunocompromised athymic Nude) MI was induced by left anterior descend-ing coronary artery (LAD) ligation upon which 5×105 cells (MSC-Luciferase-GFP, MSC-SDF-1-Luciferase-GFP and MSC-HO-1-Luciferase-GFP) were injected in 4 sites of infarct border zone (2.5 µl each, 10 µl in total). In pigs, MI was induced by temporary occlusion of LAD with balloon catheter. Subsequently, after 2 weeks, 8×106 cells (MSC-Luciferase-GFP and MSC-HO-1-Luciferase-GFP) were administered intracoronary with microcatherer. Results: Echocardiographic evaluation of heart function performed 7, 14, 28 and 42 days upon MI induction in mice indicated no therapeutic effect of genetically modified MSC. Simi-larly, echocardiographic analysis of ejection fraction in pigs four weeks upon cells administration revealed no therapeu-tic effect of either MSC-Luciferase-GFP and MSC-HO-1-Luciferase-GFP. Conclusions: Our study did not con-firm therapeutic benefits of genetically modified MSC in both acute and chronic model of myocardial damage.

Acknowledgements:The study was supported by The National Centre for Research and Development via STRATEGMED programme (STRATEGMED 2/269415/11/NCBR/2015).

66 Session 11: Medical Biotechnology in Regenerative Medicine and Drug Research


O11.3

Alpha ketoglutarate (AKG) induces apoptosis in osteosarcoma cells in vitro through the activation of JNK MAP kinaseKatarzyna Kaławaj1, Adrianna Sławińska-Brych2, Magdalena Mizerska-Kowalska1, Aleksandra Żurek1, Agnieszka Bojarska-Junak3, Martyna Kandefer-Szerszeń1, Barbara Zdzisińska1

1Department of Virology and Immunology, Maria Curie-Sklodowska University, Lublin, Poland; 2Department of Cell Biology, Maria Curie-Sklodowska University, Lublin, Poland; 3Chair and Department of Clinical Immunology, Medical University of Lublin, Lublin, PolandKatarzyna Kaławaj <[email protected]>

AKG is a key metabolite in the Krebs cycle. It has been shown that exogenous AKG exerts anti-tumour activity by reducing the level of the HIF-1α subunit and inhibiting angiogenesis in hypoxic conditions [1, 2]. Moreover, it in-hibited proliferation of colon adenocarcinoma cells in vitro in normoxic conditions [3]. The aim of this study was to evaluate the anticancer activity of AKG in human osteosar-coma cell lines, Saos-2 and HOS, and identify the mecha-nisms involved in this action. Anti-proliferative activity of AKG was estimated by the BrdU assay. An Annexin V-FITC/PI double labelling was used to detect apoptosis. The expression of apoptosis-associated proteins such as caspase-9 and Bax was determined by Western blot analy-sis, while caspase-3 activity was assessed by flow cytometry technique. The phospho- and total MAP kinases (JNK, ERK1/2) levels were measured by the ELISA method. The results showed that AKG inhibited proliferation of both osteosarcoma cell lines in concentration-dependent man-ner. It suppressed the activity of ERK 1/2 and enhanced phosphorylation of JNK kinases, increased the Bax pro-tein level, and activated caspases -9, and -3 in osteosarcoma cells. SP600125, the selective inhibitor of JNK, abolished the effect of AKG on induction apoptosis and phospho-rylation of JNK. Our results demonstrated that AKG in-duces intrinsic apoptotic pathway in osteosarcoma cells via JNK activation. Therefore, it is suggested that AKG may be used as an anti-cancer agent in the treatment of osteo-sarcoma.References:1. Matsumoto K et al. (2006) J Cell Physiol 209: 333-340.2. Matsumoto K et al. (2009) Cancer Sci 100: 1639-1647.3. Rzeski W et al. (2012) Scand J Gastroenterol 47: 565-571.Acknowledgments:This study was supported by research Grant No 2013/11/B/NZ4/04557 from State Funds for Scientific Research National Science Centre (NCN), Poland.

O11.4

3-Bromopyruvic acid: a small molecule with strong anti-tumor potencyIzabela Sadowska-Bartosz1, Grzegorz Bartosz1,2

1Department of Analytical Biochemistry, Faculty of Biology and Agriculture, University of Rzeszów, Zelwerowicza Street 4, 35-601 Rzeszów, Poland; 2Department of Molecular Biophysics, Faculty of Biology and Environmental Protection, University of Łódź, Pomorska Street 141/143, 90-236 Łódź, PolandIzabela A. Sadowska-Bartosz <[email protected]>

3-Bromopyruvic acid (3-BP), an analog of pyruvate and lactate, can “trick” cancer cells and enter as a Trojan horse. We characterized the kinetics of 3-BP transport into eryth-rocytes and demonstrated competitive inhibition of its transport by pyruvate. Flavonoids, such as luteolin and quercetin, also inhibited 3-BP transport. We found that 3-BP induces oxidative stress in erythrocytes and human non-invasive MCF-7 and invasive MDA-MB-231 breast cancer cells via depletion of glutathione, inactivation of antioxidant enzymes and provoking transient increase in the production of reactive oxygen and nitrogen species. Our studies showed that glutathione depletion is mainly due to the formation of a 3-BP conjugate (pyruvylo-S-glu-tathione). 3-BP can sensitize to cytostatic drugs cells show-ing multidrug resistance dependent on overexpression of ABC transporters since ATP depletion inhibits active ex-port of chemotherapeutics. Cells overexpressing ABCB1 transporter are hypersensitive to 3-BP even in the absence of cytostatic drugs, apparently due to secondary alterations induced by overexpression of this protein. While most studies concentrate on short-term cellular effects of 3-BP, long-term effects of exposure of this compound seem to be of crucial importance for the fate of treated cells. We found that 3-BP induces changes in the expression of a small number of genes, mostly coding for antioxidant pro-teins, i. a. metallothioneins and low molecular weight thiol homeostasis enzymes.




Posters

P11.1

Alpha ketoglutarate promotes differentiation and mineralization of osteoblasts in vitroAleksandra Żurek1, Magdalena Mizerska-Kowalska1, Adrianna Sławińska-Brych2, Katarzyna Kaławaj1, Agnieszka Bojarska-Junak3, Martyna Kandefer-Szerszeń1, Barbara Zdzisińska1

1Department of Virology and Immunology, Maria Curie-Sklodowska University, Lublin, Poland; 2Department of Cell Biology, Maria Curie-Sklodowska University, Lublin, Poland; 3Chair and Department of Clinical Immunology, Medical University of Lublin, Lublin, PolandMagdalena Mizerska-Kowalska <[email protected]>

Alpha ketoglutarate (AKG), the key intermediate in the Krebs cycle, is a multifunctional endogenous metabolite involved in different metabolic and cellular pathways. In re-cent years, numerous in vivo studies have shown that AKG supplementation may have a beneficial effect on bone tis-sue. The purpose of our study was to assess the impact of AKG on osteogenesis in vitro. The human and mouse pre-osteoblastic cell lines, hFOB 1.19 and MC3T3-E1, were used in our studies. Cell proliferation was measured by BrdU assay. ALPL, COL1A1, IBSP, SPP1, BGLAP, Runx2 and SP7 mRNA expression was analyzed by qRT-PCR. pNPP was used as a substrate to quantify ALP ac-tivity in cell cultures using colorimetric assay. The type I collagen level was determined by Western blot analysis. The BSPII, OPN, and OCN levels were measured in cell culture supernatants with the ELISA method. The degree of extracellular matrix mineralization was determined us-ing Alizarin Red S staining. The results showed that AKG did not increase the proliferation of osteoblasts. However, it enhanced the mRNA and protein levels of differentia-tion markers including ALP, COL1A1, BSPII, OPN, and OCN as well as the levels of mineralization in both cell lines. Moreover, it enhanced the mRNA expression of RUNX2 and Osterix, the key osteogenesis-related genes. We can conclude that AKG might be a promising candidate for bone anabolic drug in the prevention or treatment of osteoporosis.Acknowledgments:This work was supported by research Grant No 2013/11/B/NZ4/04557 from State Funds for Scientific Research National Science Centre (NCN), Poland.

P11.2

Small-molecule inhibition of PEX5-PEX14 protein-protein interactionM. Kolonko1,2, M. Dawidowski2,3, V. C. Kalel4, R. Erdmann4, M. Sattler2,3, G. M. Popowicz2,3

1Wrocław University of Science and Technology, Faculty of Chemistry, Department of Biochemistry, Wrocław, Poland; 2Institute of Structural Biology, Helmholtz Zentrum München, Germany; 3Center for Integrated Protein Science Munich at Chair of Biomolecular NMR, Department Chemie, Technische Universität München, Germany; 4Institute of Biochemistry and Pathobiochemistry, Department of Systems Biochemistry, Faculty of Medicine, Germany.Marta Kolonko <[email protected]>

The Trypanosoma parasites infect humans and domestic mammals, causing significant mortality and huge economic losses. African sleeping sickens (HAT) is an example of life threatening trypanosomiasis. It is caused by Trypanosoma brucei. While recent clinical trials provide hope for more ef-ficient cure for HAT other trypanosomiases require medi-cation that have serious side effects, require long treatment period and often fail to cure parasitemia. That is why new therapeutic strategies are needed.Trypanosoma protists carry out glucose metabolism in or-ganelle called glycosomes. The biogenesis of glycosomes depends on the PEX5/ PEX14 protein-protein interaction (PPI). It is essential for the transport of glycosomal en-zymes (recognized and bound by PEX5 receptor) from the cytoplasm into the organelle. It was presumed that disrup-tion of the PEX5/PEX14 complex formation results in mislocalisation of enzymes, causing metabolic collapse and it kills the parasite.We have reported a way to selectively kill T. brucei. The PEX14 has been identified as an “Achilles’ heel” of the parasite suitable for the development of new therapies. We used our nuclear magnetic resonance (NMR) and high-resolution X-ray structural information of PEX5-PEX14 complex, to design small molecules that efficiently disrupt the interaction. These molecules have trypanocidal activi-ties comparable to approved medications and are active in vivo. We are currently optimizing our PEX14 inhibitors for better ADME parameters. This is essential for optimal in vivo efficacy.




P11.3Improved production of caffeoylquinic acid derivatives in pRi-transformed plants of Rhaponticum carthamoides and biological activityEwa Skała1, Laurent Picot2, Michał Bijak3, Joanna Saluk-Bijak3, Tomasz Kowalczyk4, Janusz Szemraj5, Agnieszka Kicel6, Monika A. Olszewska6, Przemysław Sitarek1

1Medical University of Lodz, Department of Biology and Pharmaceutical Botany, Łódź, Poland; 2University of La Rochelle, UMRi CNRS7266 LIENSs, France; 3University of Lodz, Department of General Biochemistry, Faculty of Biology and Environmental Protection, Łódź, Poland; 4University of Lodz, Department of Genetics and Plant Molecular Biology and Biotechnology, Łódź, Poland; 5Medical University of Lodz, Department of Medical Biochemistry, Łódź, Poland; 6Medical University of Lodz, Department of Pharmacognosy, Łódź, PolandEwa Skała <[email protected]>

Rhaponticum carthamoides (Willd.) Iljin of the family Aster-aceae, is an endemic, perennial plant found in the natural habitat in South Siberia, Central Asia and China [1]. R. carthamoides roots and rhizomes are valued in traditional Si-berian medicine for the treatment of weakness after illness, and are used in supplements as an anabolic and adaptogen-ic and are thought to alleviate physical weakness and mental weariness [1]. Phytochemical studies indicate that this plant contains various classes of secondary metabolites such as ecdysteroids, phenolic acids with caffeoylquinic acid de-rivatives, flavonoids, polyacetylenes, sesquiterpene lactones and triterpenoid glycosides [1, 2].Transformed roots of R. carthamoides have previously been obtained by the transformation with Agrobacterium rhizo-genes [2]. Such transformed roots may represent an efficient route of obtaining valuable plant materials, a relevant al-ternative to plants cultivation in the field, especially when the raw materials are the roots: this approach avoids the destruction of the whole plant. The aim of this study was to obtain whole transformed R. carthamoides plants from shoots regenerated from transformed roots, to perform a chemical analysis of plant materials, to investigate the an-tioxidant activity of the aqueous methanol extracts from transformed plants on human blood plasma and to assess their cytotoxic activity on the viability of the A2058 human melanoma cell line.The transformed roots of R. carthamoides were able to spon-taneously regenerate green buds and shoots. Bud and shoot regeneration was observed in more than 90% of the cul-tures grown in Schenk and Hildebrandt (SH) or Gamborg (B5) liquid medium with full micro- and macronutrient concentrations, under light conditions. The regeneration of shoots from transformed roots may be improved by sup-plementation of Murashige and Skoog (MS) agar medium with indole-3-acetic acid (IAA) and benzyladenine (BA). The shoots were multiplied on MS medium supplemented with 0.1 mg×L-1 IAA and 0.2 mg×L-1 BA and then rooted successfully. The transformation of the plants derived from transformed roots was confirmed by PCR analysis and Southern hybridization. The three-month-old transformed plants (TR-plants) were able to biosynthesize caffeoylquinic acid derivatives such as chlorogenic acid, 3,5- or 4,5-di-O-caffeoylquinic acids. The transformed plants demonstrated higher secondary metabolite levels and greater biomass ac-cumulation than the plants obtained directly from seeds (SD-plants) of the same age and grown under the same condi-tions as transformed plants. It was found that extracts from TR-plants inhibited the growth of A2058 human melanoma cells depending on the tested concentration and bestowed antioxidant properties on human blood plasma.References:Kokoska L, Janowska D (2009) Phytochemistry 70: 842-855.Skała E et al. (2015) BioMed Res Int. Article ID 181098: 1-11.

P11.4

Growth of Leonurus sibiricus L. roots with over-expression of AtPAP1 transcriptional factor in closed bioreactor, production of bioactive phenolic compounds and evaluation of their biological activityPrzemysław Sitarek1, Tomasz Kowalczyk2, Laurent Picot3, Dorota Michalska-Hejduk4 , Michał Bijak5, Adam J. Białas6, Marzena Wielanek7, Tomasz Śliwiński8, Ewa Skała1

1Medical University of Lodz, Department of Biology and Pharmaceutical Botany, Łódź, Poland; 2University of Lodz, Department of Genetics and Plant Molecular Biology and Biotechnology, Łódź, Poland; 3Université de la Rochelle, UMRi CNRS 7266 LIENSs, France; 4University of Lodz, Department of Geobotany and Plant Ecology, Faculty of Biology and Environmental Protection, Łódź, Poland; 5University of Lodz, Department of General Biochemistry, Faculty of Biology and Environmental Protection, Łódź, Poland; 6Medical University of Lodz, Department of Pneumology and Allergy, 1st Chair of Internal Medicine, Łódź, Poland; 7University of Lodz, Department of Plant Physiology and Biochemistry, Faculty of Biology and Environmental Protection, Łódź, Poland; 8University of Lodz, Laboratory of Molecular Genetics, Łódź, PolandPrzemysław Sitarek <[email protected]>

Leonurus sibiricus L. belongs to the Lamiaceae family. It is an aromatic, herbaceous plant, which can be annual, bisannual or perennial. Most significantly from a pharmacological perspective, the species has been reported to have a wide range of anti-inflammatory, antioxidant, anti-diabetes, anti-bronchitis and anti-cancer effects (1, 2). Biological manipu-lation showed that, fast-growing transgenic root cultures of Leonurus sibiricus with the AtPAP1 transcriptional factor may be used as a base for potential large-scale phenolic ac-ids production. The present study investigates the effect of different volumes of medium (300 mL, 1 L, 3 L, 5L flasks) and a 5 L bioreactor on biomass increase and phe-nolic acids production by transgenic roots of L. sibiricus. Of these cultures, those from the 5 L bioreactor dem-onstrated the greatest increase in dry weight (20.83 g/L) and highest yields of phenolic acids (chlorogenic acid 448 mg/L and caffeic acid 302 mg/L). Additionally, they also displayed a cytotoxic effect on melanoma cells across the range of tested concentrations, as well as antioxidant activ-ity on human blood plasma. This approach may serve as an alternative to conventional field crops for enabling large-scale production of the active constituents of AtPAP1 root extract, which would be of great value for pharmaceutical production.References:Sayed A et al. (2016) Asian Pac J Trop Biomed 6: 1076-1080.Sitarek P et al. (2016) Tumour Biol 37: 8753-8764.



P11.5

Peptide‐based biopolymers PolyRGD to be used for pro-regenerative purposesAgnieszka Żylicz-Stachula1,2, Joanna Żebrowska1,2, Natalia Krawczun1,2, Małgorzata Palczewska1, Piotr Mucha3, Piotr M. Skowron1,2

1University of Gdansk, Faculty of Chemistry, Department of Molecular Biotechnology, Gdańsk, Poland; 2BioVentures Institute Ltd., Poland; 3University of Gdansk, Faculty of Chemistry, Department of Molecular Biochemistry, Gdańsk, PolandAgnieszka Żylicz-Stachula <[email protected]>

The RGD motif (Arg-Gly-Asp) has been selected for po-ly-epitope/poly-signal protein construction. This motif is common for cell-secreted extracellular matrix molecules (ECM). Activity of the RGD sequence exhibits mainly dur-ing the extracellular signal transduction and cell prolifera-tion. The PolyRGD proteins can find applications in the re-generation of cells, tissues and organs. Placed in a wound, they can act directly or gradually release the biologically ac-tive peptides, due to their autodegradation or activity of the extracellular matrix proteases.First, monomeric peptide units for construction of poly-peptide protein structures were selected and the PolyRGD proteins were designed. Then, a series of DNA vectors was constructed, enabling amplification of the selected epitopes and expression of the recombinant genes in Escherichia coli.The PolyRGD proteins were obtained using: (i) bioinfor-matic methods for the design of the genes, (ii) chemical synthesis and molecular cloning of the designed, mono-meric DNAs units and (iii) the DNA units’ amplification. For this purpose, a patented technology was used (PL 228341). The technology involves a directional, vector-en-zymatic amplification of the designed DNA segment, with maintenance of the correct open reading frame for a given insert.The obtained recombinant genes were successfully ex-pressed in E. coli. Procedures for efficient isolation and pu-rification of the bioactive PolyRGD protein variants were developed.

P11.6

Identification of a novel protein interaction with the EKLF transcription factor (Erythroid Krüppel-like factor)Anna Witucka, Klaudia Kulczyńska, Mirosława Siatecka

Institute of Experimental Biology, Department of Genetics, University of Adam Mickiewicz, 61-614 Poznań, Poland.Miroslawa Siatecka <[email protected]>

Erythroid Krüppel-like factor (EKLF/KLF1) is a tran-scription factor that plays a critical role in activation of genes essential for the red blood cell development. It is achieved by EKLF’s ability to directly integrate basal tran-scription and chromatin-modifying machineries. EKLF in-teracts with chromatin remodeling complex SWI/SNF, the HIRA chaperon protein and components of the transcrip-tion machinery: p62 a subunit of TFIIH and TAF9. Post-translational modifications of EKLF provide alternate pro-tein interaction platforms that lead to varied downstream effects in erythroid differentiation.Our laboratory is interested in elucidation of molecular mechanism of genetic distortions resulting from the pres-ence of dominant mutations in EKLF. We investigate whether the functional differences between WT and mu-tated EKLF follow from formation of slightly different protein complexes e.g. with transcriptional machinery.In order to analyze this, we established stable K562 cell lines with doxycycline inducible expression of Flag-EKLF variants. Protein complexes were isolated by immunopre-cipitation with the ANTI-FLAG M2 magnetic beads and identified by mass spectrometry. After applying the Mas-cot program and differential comparison with negative controls approximately 100 EKLF specific partners were selected. A new EKLF’s interaction with USP7 (Ubiquitin carboxyl-terminal hydrolase 7) was empirically confirmed.Acknowledgements:Financial support: OPUS grant 2013/09/B/NZ1/01879 of Polish Na-tional Science Center.



P11.7

Cytotoxic potential analysis of biomaterials modified with proteolytic enzyme inhibitorsKatarzyna Szałapata1, Mateusz Pięt2, Justyna Kapral3, Roman Paduch2, Bożena Pawlikowska-Pawlęga3, Monika Osińska-Jaroszuk1, Anna Jarosz-Wilkołazka1

1Department of Biochemistry, Faculty of Biology and Biotechnology, Maria Curie-Sklodowska University, Lublin, Poland; 2Department of Virology and Immunology, Faculty of Biology and Biotechnology, Maria Curie-Sklodowska University, Lublin, Poland; 3Department of Comparative Anatomy and Anthropology, Faculty of Biology and Biotechnology, Maria Curie-Sklodowska University, Lublin, PolandKatarzyna Szałapata <[email protected]>

The formation of infections within the prosthesis implant-ed into the human body is a serious problem for modern medicine. Currently, many new solutions are sought in the field of biomaterial engineering, thanks to which it is possible to increase the aseptic potential of various types of biomedical materials and to give them antimicrobial properties. One of the innovative solutions is the use of proteolytic enzyme inhibitors (synthetic inhibitor AEBSF and α1-antitrypsin) as agents preventing the adhesion of pathogenic microorganisms (such as E. coli, P. aeruginosa, S. aureus or C. albicans) to the surface of prostheses and inhibiting the formation of biofilm produced by these mi-croorganisms. During the development of new biomateri-als, their possible cytotoxicity to the tissues of the human body should be tested.Our study investigates the influence of vascular prosthe-ses modified with two proteolytic enzyme inhibitors on the growth of human skin fibroblasts (HSF). The LDH (lactate dehydrogenase) cytotoxicity analysis of the modified bio-materials revealed that the Uni-Graft prosthesis modified with AEBSF inhibitor and α1-antitrypsin does not show cy-totoxic effect on human epithelial cells (HSF). The results of the LDH test were also confirmed by microscopic pho-tographs on which no abnormalities in the morphological structure of HSF cells were observed.Acknowledgments:This work was partially supported by National Science Center (2014/15/N/NZ7/04092 and 2017/25/B/NZ7/01084).

P11.8

Functionalization of silk with metal binding peptides to produce iron oxide/silk composite spheres for theranostics purposeKamil Kucharczyk1,2, Tomasz Deptuch1,2, Karolina Penderecka1,2, Andrzej Mackiewicz1,2, Hanna Dams-Kozlowska1,2

1Poznan University of Medical Sciences, Department of Medical Biotechnology, Poznań, Poland; 2Greater Poland Cancer Centre, Department of Diagnostics and Cancer Immunology, Poznań, PolandHanna Dams-Kozlowska <[email protected]>

Spider silk is a biocompatible and biodegradable material with broad biomedical applications. It can be processed into different morphological forms including micro- and nanospheres. Moreover, it can be genetically engineered to obtain material of modified properties. Previously we characterized the EMS2/IONPs spheres that were com-posed of bioengineered silk EMS2 and iron oxide nanopar-ticles (IONPs). The drug loading studies exhibited more than two-fold higher loading efficiency of doxorubicin to spheres EMS2/IONPs than to EMS2. To further increase the affinity of silk to IONPs, we functionalized silk with metal binding peptides Fe1 and Fe2.The study aimed to develop iron oxide/silk composite ma-terial for the theranostic application.The functionalized (MS1Fe1)2, (MS1Fe2)2 and MS-1Fe1MS1Fe2 and control sMS1 silks were produced. The functionalizations did not impede the self-assembly of silk, and spheres were obtained. The SEM/EDS analysis of the composite silk/IONPs spheres indicated that the func-tionalized silks bound more IONPs comparing with the control silks (sMS1 and EMS2). Moreover, the Fe1 peptide bounded more IONPs than the Fe2 peptide. The binding of IONPs of negative charge resulted in the formation of spheres with more separated and more spherical morphol-ogy than spheres with positive IONPs. The spheres with a higher IONPs content may result in higher loading effi-ciency of doxorubicin, which may be beneficial for medical applications.



P11.9

How to create drug delivery systems based on supramolecular compounds, proteins and carbon nanotubes?A. Jagusiak, B. Piekarska, B. Stopa, G. Zemanek, K. Chłopaś, O. Chmura

Jagiellonian University Medical College, Faculty of Medicine, Chair of Medical Biochemistry, Kraków, PolandAnna Jagusiak <[email protected]>

Construction of high capacity drug-delivery systems pre-senting tissue specificity that allows to avoid side effects is nowadays one of the most important research topics. Con-go red (CR) type supramolecular compounds (SC) were previously shown to interact with some proteins, includ-ing albumin (BSA) and antigen-bound antibodies (Ag-Ab), and can also bind to and disperse carbon nanotubes. The presented results concern the studies on the complexes between SC with anticancer drugs: doxorubicin (DOX) or imatinib (IM) and the interaction of these complexes with antibodies (Ab), albumin and carbon nanotubes (CNT).Hypothesis: SC-drug complexes interact with CNT and with proteins and can potentially deliver drugs to areas af-fected by pathological processes.• SC associate in water solutions creating ribbon-like mul-

timolecular assemblies• organic molecules (such as DOX and IM) can intercalate

into SC assemblies• the structure and protein-binding properties of CR-

DOX significantly differs from CRGoals: Description of the properties of complexes formed by SC-DOX and SC-IM with Ag-Ab, BSA, CNT and CNT-linked proteins.Research methods: electrophoresis, gel filtration, cell cul-tures, fluorescence microscopy, DLS.Results: Using BSA increase capacity for binding drugs while the application of Ab enables targeted drug delivery. The presented system offers a wide range of biomedical applications including drugs delivery to cancer cells. As carriers of drugs, these complexes present an interesting alternative to the currently used systems.References: Jagusiak A et al. (2017) Beilstein J Nanotechnol 8: 636-648.Roterman I, Konieczny L (2017) Springer Open Self-Assembled Molecules – New Kind of Protein Ligands.Acknowledgements:We acknowledge the financial support from the National Science Centre, Poland (grant no. 2016/21/D/NZ1/02763).

P11.10

Tetragametic chimeras in DNA testing, diagnostics, and therapyAnna Jędrzejak

Regenerative Medicine and Cancer Research Section, Student's Naturalists Science Club, Adam Mickiewicz University in Poznań, Poznań, PolandAnna Jędrzejak <[email protected]>

Tetragametic chimeras are created by the fusion of two zy-gotes, typically at the very early stage of dizygotic pregnan-cy. The result is an organism containing two cell popula-tions, carrying distinct DNA sets and distributed randomly within the body. For a long time medicine did not realize their existence and its consequences. Secondary cell popu-lation can occur within the pigmented tissues and manifest itself by color difference, allowing easy identification - but if it localizes within the internal organs, it becomes barely detectable. There is still no certain information about the rate of the occurrence of the tetragametic chimerism. As during the zygote fusion cells segregate randomly, there is no way to predict which tissues can have different DNA set than the rest of the individual. Up to now all known cases of tetragametic chimerism were diagnosed during unrelat-ed medical procedures such as transplantation or parent-age testing. To increase chances of identifying a chimera, the samples for DNA test would have to be taken from multiple tissues, including internal organs, which would be very invasive and costly procedure. Moreover, such analysis could be insufficient to confirm a disease of genetic back-ground in patients, as the cause of the disease would re-main unknown because the genetic change occurred in the different cell population. It could also potentially obstruct the treatment due to problems with selective drug delivery.



P11.11

Endocytosis, intracellular trafficking and degradation behavior of functionalized silk particles for targeted drug deliveryAnna Florczak1,2, Andrzej Mackiewicz1,2, Hanna Dams-Kozlowska1,2

1Chair of Medical Biotechnology, Poznan University of Medical Sciences, Poznań, Poland; 2Department of Diagnostics and Cancer Immunology, Greater Poland Cancer Centre, Poznań, PolandAnna Florczak <[email protected]>

One of strategies for cancer treatment is to design a nan-odelivery system, directing active agents specifically to the site of tumor, therefore avoiding potential side effects. The bioengineered spider silk is a material with a great potential in biomedical application. Drug-encapsulated biodegrad-able spheres made of functionalized silk have considerable potential for use as delivery systems. Inclusion of targeting ligands displayed at the surface of a carrier, with specific affinity towards a particular receptor, can further enhance the accumulation and uptake of these nanoparticles at the site of action.The objective was to investigate the mechanism involved in the internalization pathway into target cells, intracellular trafficking and biodegradation of the obtained spheres in live cells.The hybrid constructs were obtained by adding a Her2-binding peptide (H2.1) to MS1 and MS2 bioengineered silks based on the MaSp1 and MaSp2 proteins from N. clavipes, respectively. The H2.1MS1 and H2.1MS2 proteins were blended at a weight ratio of 8:2. Stable silk parti-cles were formed by mixing a soluble protein with potas-sium phosphate using a micromixing technique. Obtained spheres were characterized in terms of size and morphol-ogy using an SEM. The uptake of spheres by human Her2-positive breast cancer cells SKBR3 in the presence of en-docytosis inhibitors was assessed using flow cytometry. The subcellular distribution of silk particles was investigated using CLSM by evaluating the signal colocalization with organelle-specific tracker. CLSM imaging was performed in order to analyze the degradation kinetics of silk particles by employing different lysosomal and exosomal inhibitors.Functionalized spheres were actively internalized by Her2-positive cells. Silk particles facilitated the entry into cells both through the clathrin- and caveolae-dependent path-way of endocytosis. Silk spheres upon entering the cells localized in lysosomes. Applying various lysosomal inhibi-tors substantially extended the fluorescence signal from silk particles indicating the intracellular degradation took place in lysosomes. Furthermore, the prolonged presence of silk particles was not observed when using inhibitors of exoso-mal release, which further proved that the lysosomal func-tion is essential for silk-based carriers elimination.We showed for the first time, that silk spheres could be processed into cells by the endocytosis pathway and their degradation occurred in lysosomes. The degradation of the carrier is of a great importance to develop drug delivery system.

P11.12

Genistein-mediated lysosomal degradation of main patogenic factors of Alzheimer’s disease as a novel therapeutic strategyKarolina Pierzynowska1, Magdalena Podlacha1,2, Lidia Gaffke1, Irena Majkutewicz2, Jagoda Mantej1, Dorota Myślińska2, Grzegorz Węgrzyn1

1Department of Molecular Biology, Faculty of Biology, University of Gdańsk, Wita Stwosza 59, 80-308 Gdańsk, Poland; 2Department of Animal and Human Physiology, Faculty of Biology, University of Gdańsk, Wita Stwosza 59, 80-308 Gdańsk, PolandKarolina Pierzynowska <[email protected]>

Alzheimer’s disease (AD) is one of the most severe neu-rological diseases, and the number of patients is growing rapidly. Unfortunatelly, no effective treatment is currently available despite many therapeutic strategies. The most re-cent studies indicated that the main pathogenic factor of AD – β-amyloid can be degradated by lysosomes, thus its accelerated lysosomal degradation became a promissing strategy for treatment of AD. However, search for the safe activator of lysosomes is still ongoing. Our previous studies indicated that genistein, one of isoflavones, can stimulate biogenesis of lysosomes and activity of acid hydrolases by positive regulation of trancription factor EB gene expres-sion. Thus, the aim of this study was to determine thera-peutic effects of genistein on AD. The streptozotocin rat model of this disease was used. Behavioral studies showed that genistein corrected locomotor activity of AD rats (ac-tometers, open field), alleviated stress (elevated plus maze) and improved memory (Morris water maze), making them indistinguishable from healthy rats. Biochemical assays indi-cated that genistein decreased levels of both β-amyloid and hyperphosphorylated tau protein in the brain to the level of these proteins in healthy rats (Western blotting, immu-nohistochemistry) while cell culture experiments showed that this effect required lysosomes. In summary, genistein as a safe compound which can be used in long-term treat-ment has a great chance to become a cure for AD.



P11.13

Degradation of glycosaminoglycans through autophagy stimulation in mucopolysaccharidosis type IIILidia Gaffke, Karolina Pierzynowska, Grzegorz Węgrzyn

University of Gdańsk, Department of Molecular Biology, Gdańsk, PolandLidia Gaffke <[email protected]>

Mucopolysaccharidoses (MPS) belong to lysosomal storage diseases which are caused by lack or decresed activity of enzymes involved in the degradation of unbranched chains of polysaccharides called glycosaminoglycans (GAGs). Normally, GAGs degradation takes place in lysosomes due to the activity of several enzymes that cooperate to remove individual mono-sugars or chemical groups from GAG chains. Defect of one of the enzymes prevents the activity of the others because of sequential way of degradation. Therefore, GAGs degradation stops at some point, and un-degraded molecules accumulate in the lysosomes.Our previous studies shown, that one of the currently test-ed therapeutics – genistein – can lower the level of GAGs storage in cells by increasing the biogenesis of lysosomes, thus we argue that in the autophagy process accumulated GAGs can be degradated as well. Autophagy, induced by various natural compounds, is considered one of the most promising therapeutic strategies in many diseases related to accumulation of various molecules. Our approach rely on testing various compounds, known to induce autophagy through different pathways. We found that genistein, treha-lose, curcumin, capsaicin, resveratrol, and calcitriol differ-entially affected levels of GAGs in MPS III cells. Further investigation reaveld additive affect after combine genistein with different compounds. These results are considered as possible use of the tested compounds in potential therapy for MPS III.

P11.14

Structural investigations on the components synergy of a new proposed regenerative treatment for periodontal lesionsEugenia Eftimie Totu1, Roxana Buga1, Daniel Petre1, Tiberiu Totu1, Daniela Mănuc2, Corina Marilena Cristache2

1University Politehnica of Bucharest, Romania; 2University of Medicine and Pharmacy Carol Davila, Bucharest, RomaniaEugenia Eftimie Totu <[email protected]>

In the current clinical activity, it is used a paste contain-ing tetracycline, metronidazole and white vaseline for peri-odontal wounds treatment, but the beneficial effect of this treatment is not complete satisfactory. Therefore, we pro-pose a new topic treatment material, coupling the antimi-crobial effects of antibiotics with the specific proliferation and differentiation of osteoblasts activity of melatonin and hyaluronic acid while inhibiting the osteoclasts activ-ity. The new material is proposed for directly usage in the periodontal pockets. During last years, both melatonin and hyaluronic acid were studied due to their beneficial effects in medical area. It has been proved that melatonin inter-feres in the osteoclasts function thus inhibiting bone re-sorption, due to its antioxidant properties. The hyaluronic acid showed anti-inflammatory and anti-bacterial effects in the treatment of periodontal diseases, presenting specific tissue healing properties. The present work aims to develop a derivative UV spectrophotometric method for simultane-ous estimation of melatonin, hyaluronic acid and antibiotic in combined dosage form. Absorbance of all components within the new treatment were measured at specific λ max. It has been proved that all components in treatment mixture preserve their specific activity.AcknowledgementThis work was supported by a grant of the Romanian National Author-ity for Scientific Research and Innovation, CCCDI – UEFISCDI, project number 39/2018 COFUND-MANUNET III-HAMELDENT, within PNCDI III.



P11.15

3-Bromopyruvic acid affects the mobility and redox balance of metastatic cellsMonika Pichla1, Natalia Pieńkowska1, Jolanta Sroka2, Katarzyna Piwowarczyk2, Zbigniew Madeja2, Grzegorz Bartosz3, Izabela Sadowska-Bartosz1

1Department of Analytical Biochemistry, Faculty of Biology and Agriculture, University of Rzeszów, Zelwerowicza 4, 35-601 Rzeszów, Poland; 2Department of Cell Biology, Jagiellonian University, Gronostajowa 7, 30-387 Cracow, Poland; 3Department of Molecular Biophysics, University of Łódź, Pomorska 141/143, 90-236 Łódź, PolandGrzegorz Bartosz <[email protected]>

Cancer treatment becomes very difficult when tumor cells metastasize into distant body organs. Therefore, preven-tion of cancer cell metastasis is an effective strategy for successful management of human cancers. An alkylating antiglycolytic compound, 3-bromopyruvic acid (3-BP) is a promising anticancer agent, able to act also on multidrug-resistant cells in vitro. We have studied the effect of 3-BP on the survival, mobility, ATP content and glutathione content of human prostate metastatic carcinoma DU-145 cells. The cells were sensitive to 3-BP action, IC50 estimated with Neutral Red after 24 h being 25.8±1.1 µM. The cell mobility was strongly affected by 3-BP, total length of cell displacement being decreased from 100.9±7.3 down to 78.6±6.7 µm during 6 h, and the rate of cell movement being reduced from 0.28±0.02 to 0.22±0.02 µm/min, 24 h after treatment with 10 µM 3-BP (P<0.05). The inhibi-tion of cell movement can be attributed to ATP depletion, which decreased to 9.9±5.6% of the control value at 30 µM 3-BP (P<0.05). 3-BP brought about changes in the level of glutathione, increasing glutathione content at low concen-trations and decreasing it at higher concentrations (30–50 µM), thus altering the redox equilibrium of the cells. The high sensitivity of metastatic DU-145 cells to the action of 3-BP and inhibition of their motility by low concentrations of this compound suggest that 3-BP may prove useful in the therapy of metastatic tumors.

P11.16

Nrf2 influences satellite cells and muscle regeneration but does not aggravate the progression of Duchenne muscular dystrophyIwona Bronisz-Budzynska, Magdalena Kozakowska, Katarzyna Chwalenia, Paulina Podkalicka, Olga Mucha, Agnieszka Loboda, Alicja Jozkowicz, Jozef Dulak

Jagiellonian University, Faculty of Biochemistry, Biophysics and Biotechnology, Department of Medical Biotechnology, Kraków, PolandIwona Bronisz-Budzyńska <[email protected]>

The nuclear factor (erythroid-derived-2)-like 2 (Nrf2) is a transcription factor known as a master regulator of the inflammatory response. Our aim is to assess the Nrf2 in-fluence on myoblast and muscle satellite cells (mSCs) dif-ferentiation and its role in the progression of Duchenne muscular dystrophy (DMD).In order to evaluate the role of Nrf2 in muscle satellite cells differentiation, mSCs were isolated from Nrf2+/+ and Nrf2-/- mice and in vitro differentiation was performed in an environment of different oxygen concentration. Sat-ellite cells lacking Nrf2 demonstrated more robust differen-tiation in hypoxic condition. Interestingly, their growth and viability were strongly impaired in normoxia in comparison to wild type cells.To investigate the role of Nrf2 in the progression of the Duchenne muscular dystrophy double knockouts were generated. Firstly, mice were subjected to an exhaustion treadmill test to assess muscle functionality. Animals’ per-formance demonstrated that lack of Nrf2 does not affect exercises capacity. This observation was validated by histo-logical and FACS analysis showing a similar level of muscle damage, as evidenced by the degree in inflammatory cells infiltration and fibrosis in dystrophin-deficient mice (mdx) and double knockouts. In summary, lack of Nrf2 activity in mSCs of mdx mice enhanced the mSCs differentiation in the hypoxia condi-tion. However, this does not aggravate the progression of Duchenne muscular dystrophy.



P11.17

The influence of polyphenolic phytochemicals on the redox homeostasis of cellsMonika Baranowska, Klaudia Suliborska, Zuzanna Koziara, Emon Asaduzzaman, Wojciech Chrzanowski, Jacek Namieśnik, Agnieszka Bartoszek

Gdansk University of Technology, Faculty of Chemistry, Gdańsk, PolandMonika Baranowska <[email protected]>

The enhancement of endogenous antioxidant defense sys-tem through dietary supplementation appears to be a rea-sonable approach to reduce oxidative stress. However, to pinpoint the actual role of dietary antioxidants in maintain-ing cellular redox homeostasis, the relation between their physicochemical, electrochemical and biological properties must be established. The aim of our study was to clarify these relationships for selected polyphenoles as well as to reveal molecular implications resulting from exposure to the tested compounds for oxidative stress response system in HT29 cell line.Reduction potentials of phenolic compounds were de-termined by potentiometric titration. The biological tests embraced determinations of cytotoxicity and antioxidant activity in cell culture assessed by CAA assay. Genomic studies concerning modulation of expression of genes involved in oxidative stress response employed RT-PCR array-based technologies.Our comparative approach to establish the correlation be-tween chemical properties and biological potential of poly-phenols suggests their dose-dependent role in maintenance of redox homeostasis. This influence was accompanied with modulated expression of genes involved in oxidative stress response. The set of regulated genes was dependent not only on structure of investigated compounds, but also on their standard reduction potentials. The final conclusion is that dose dependent effects of dietary antioxidants and biological functions potentially modulated by them may be-come deregulated upon exposure to their excessive doses. Acknowledgement:This work is supported by the project financed by National Science Cen-tre, Poland in a programme „MAESTRO 6” (application 2014/14/A/ST4/00640).

P11.18

CRISPR/Cas9 editing of heme oxygenase-1 affects cardiomyocytes differentiated from human induced pluripotent stem cellsMateusz Jeż1, Jacek Stępniewski1, Kalina Andrysiak1, Alan Kania2, Łukasz Chrobok2, Katarzyna Palus-Chramiec2, Marian H. Lewandowski2, Alicja Józkowicz1, Józef Dulak1,3

1Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland; 2Department of Neurophysiology and Chronobiology, Institute of Zoology and Biomedical Research, Jagiellonian University, Kraków, Poland; 3Silesian Park of Medical Technology, Kardio-Med Silesia, Zabrze, Poland

Background: Human induced pluripotent stem cells (hiPSC) are an invaluable platform for generation of hu-man cardiomyocytes for disease modeling, drug screening and possibly also for regenerative medicine. Importantly, efficient and reproducible protocols for differentiation of hiPSC into cardiomyocytes (CMs) have been described, however, hiPSCs-CMs demonstrate fetal phenotype, thus further studies investigating factors involved in their matu-ration are needed.Heme oxygenase-1 (HO-1) is a cytoprotective enzyme de-grading heme into CO, Fe2+, and biliverdin. Via products of its enzymatic activity HO-1 influences plethora of phys-iological processes including stem cell differentiation.Aim: Aim of this study was to investigate the role of HO-1 in differentiation of hiPSC into CMs.Methods: hiPSCs were differentiated into CMs using small molecular inhibitors modulating Wnt pathway. CRISPR/CAS9 technique was utilized to inactive HMOX1. Lack of HO-1 protein was functionally confirmed by Western blot technique.Results: Differentiation of hiPSC to CMs was performed with very high efficiency (up to 98.8%, basing on flow cy-tometry analysis of cardio-troponin T). Importantly, patch-clamp technique revealed shortened depolarization phase in hiPSC-CMs lacking HO-1 whereas qRT-PCR analysis confirmed lower expression of ion channels in CMs de-void of HO-1.Conclusion: Observed characteristics indicate that hiPSC- CMs lacking HO-1 are less mature than their control coun-terparts suggesting an important role of HO-1 in process of CMs maturation.

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